1. Field of the Invention
This invention pertains generally to drug delivery schemes and methods for producing biologically stable protein based nanostructures, and more particularly to compositions and methods for intracellular therapeutic protein delivery based on the formation of a protein nanocapsule having a single-protein core and a thin polymeric shell cross-linked by designed peptides that can be specifically recognized and cleaved by a protease to release the protein core.
2. Description of Related Art
Intracellular delivery of exogenous proteins holds great promise in biological and medical applications in humans and animals. For example, recombinant protein therapeutics is an attractive alternative to gene or siRNA transfections and may be useful in biological and medical applications, including cell cycle regulation, cellular immunity, transcription regulation and cancer treatments.
Protein therapeutics has enormous potential for the treatment of human diseases, especially those caused by the temporary or permanent loss of a type of functional protein. For example, many cancer cells do not undergo programmed cell death because proteins in the apoptosis machinery are either defective in function or attenuated in expression. Direct delivery of active proteins to the cytosol of cells could therefore restore or replenish the functions of interest and lead to the desired cell phenotypes. Additionally, introduction of recombinant proteins that can regulate transcription can exert artificial control of gene expression levels and could lead to the reprogramming of cell fate. In comparison to gene therapy, which is currently the predominant choice of delivery for promising protein therapeutics, direct protein delivery can bypass the requirement of permanent or unintended changes to the genetic makeup of the cell, and is therefore a safer therapeutic alternative.
Although protein-based drugs have had great commercial success, they still suffer from significant obstacles with in vivo efficacy, especially in the area of intracellular protein delivery. The development of intracellular protein therapeutics has been hampered by the limitations arising from the nature of proteins. These limitations include structural fragility, low serum stability and poor membrane permeability for most proteins that are negatively charged at pH 7. The poor stability and membrane impermeability of most native proteins make efficient delivery to the interior of cells difficult. For such technology to be successful, the exogenous protein needs to effectively penetrate the plasma membrane and to be efficiently released in the cytosol. Meanwhile, the biological activity of proteins should be resistant to denaturation and enzymatic degradation.
Different strategies that aim to protect protein integrity and activity as well as to aid with intracellular delivery have been explored. In spite of these efforts, no single technique has been widely applied because of the practical limitations of these approaches.
Among the various protein delivery approaches that have been pursued, are physical methods, such as electroporation and microinjection, a protein transduction domain (PTD)-mediated platform, and noncovalent methods based on a cationic carrier, such as liposomes or polymer particles.
However, each of these approaches has limitations that limit their usefulness. For example, physical methods such as microinjection can damage the target cells and the types of available in vivo purposes for the methods are limited.
Similarly, PTD based delivery systems that require a covalent attachment to the protein being delivered, either by creating a designed DNA construct or by a specific chemical conjugation, can impair the stability and activity of proteins in certain cases. Covalent approaches include genetic fusion of protein transduction domains and conjugation of polymers to free amine groups on the surface of proteins. However, these approaches often suffer from an alteration of protein activity due to modification of the protein structure.
Noncovalent based polymer carriers that encapsulate protein cargo via electrostatic assembly or hydrophilic and hydrophobic interactions have also been explored. These methods employ various materials to effectively help the protein travel into cells, albeit often suffer from instability in serum. However, it is still challenging to enhance delivery efficiency as well as to avoid the colloidal instability of the complex.
Accordingly, intracellular delivery of functional proteins has significant therapeutic implications in biological applications, including disease therapies, vaccination, and imaging. There is a need for a method for intracellular protein delivery and for a method for reliably producing a construct that is stable in serum and can readily enter the cytosol of target cells by endocytosis.
There is a particular need for an intracellular delivery system that can protect the protein cargo from denaturation and proteolysis during circulation and endocytosis; that can shield a negatively charged protein and provide an overall positive surface charge to facilitate internalization across the phospholipid membrane; and that can release the protein cargo in native form when the desired destination (i.e. the cytosol) is reached. The present invention satisfies these needs as well as others and is generally an improvement over the art.